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. 2006 Nov;80(21):10675-82.
doi: 10.1128/JVI.01015-06. Epub 2006 Aug 23.

The unique C termini of orthopoxvirus gamma interferon binding proteins are essential for ligand binding

Anthony A Nuara  1 , Hongdong Bai, Nanhai Chen, R Mark L Buller, Mark R Walter
Affiliations

The unique C termini of orthopoxvirus gamma interferon binding proteins are essential for ligand binding

Anthony A Nuara et al. J Virol. 2006 Nov.

Abstract

The orthopoxviruses ectromelia virus (ECTV) and vaccinia virus (VACV) express secreted gamma interferon binding proteins (IFN-gammaBPs) with homology to the ligand binding domains of the host's IFN-gamma receptor (IFN-gammaR1). Homology between these proteins is limited to the extracellular portions of the IFN-gammaR1 and the first approximately 200 amino acids of the IFN-gammaBPs. The remaining 60 amino acids at the C termini of the IFN-gammaBPs contain a single cysteine residue shown to be important in covalent dimerization of the secreted proteins. The function of the remaining C-terminal domain (CTD) has remained elusive, yet this region is conserved within all orthopoxvirus IFN-gammaBPs. Using a series of C-terminal deletion constructs, we have determined that the CTD is essential for IFN-gamma binding despite having no predicted homology to the IFN-gammaR1. Truncation of the ECTV IFN-gammaBP by more than two amino acid residues results in a complete loss of binding activity for both murine IFN-gamma and human IFN-gamma (hIFN-gamma), as measured by surface plasmon resonance (SPR) and bioassay. Equivalent truncation of the VACV IFN-gammaBP resulted in comparable loss of hIFN-gamma binding activity by SPR. Full-length IFN-gammaBPs were observed to form higher-ordered structures larger than the previously reported dimers. Mutants that were unable to bind IFN-gamma with high affinity in SPR experiments failed to assemble into these higher-ordered structures and migrated as dimers. We conclude that the unique CTD of orthopoxvirus IFN-gammaBPs is important for the assembly of covalent homodimers as well as the assembly of higher-ordered structures essential for IFN-gamma binding.

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Figures

FIG. 1.
FIG. 1.
Sequence alignment of ECTV and VACV IFN-γBPs. Predicted signal sequences are boxed. Individual amino acid differences between the proteins are highlighted. The receptor homologous region of the ECTV sequence extends from the signal sequence cleavage site to amino acid 211. The unique orthopoxvirus CTD is underlined. Covalent dimerization of the secreted molecules is mediated by cysteine 216 (ECTV) (6). Sequences were obtained from GenBank (ECTV accession no. AAC99563; VACV accession no. AAA48205) and aligned using CLUSTAL W.
FIG. 2.
FIG. 2.
Murine IFN-γ binding by IFN-γBP truncation mutants. (A) SPR sensorgrams of full-length and truncated IFN-γBPs binding to mIFN-γ. Expressed proteins were injected over a mIFN-γ sensor surface at a flow rate of 5 μl/min for 2 min. Sensorgrams are typical of those seen over several replicate injections. (B) SPR responses were normalized to wild-type ECTV IFN-γBP and include multiple injections over several IFN-γ surfaces of various densities. Truncation sites for both VACV and ECTV correspond to the C-terminal ending of the ECTV sequence as displayed in Fig. 1.
FIG. 3.
FIG. 3.
Human IFN-γ binding by IFN-γBP truncation mutants. (A) SPR sensorgrams of full-length and truncated IFN-γBPs binding to hIFN-γ. Expressed proteins were injected over an SA hIFN-γB sensor surface at a flow rate of 5 μl/min for 2 min. Sensorgrams are typical of those seen over several replicate injections. (B) SPR responses were normalized to wild-type ECTV IFN-γBP and include multiple injections over several IFN-γ surfaces of various densities. Truncation sites for both VACV and ECTV correspond to the C-terminal ending of the ECTV sequence as displayed in Fig. 1.
FIG. 4.
FIG. 4.
Kinetics analyses of C-terminal mutations for mIFN-γ. Alanine mutagenesis scanning was conducted over the last nine amino acid residues of the ECTV IFN-γBP. Mutants were tested for mIFN-γ binding by SPR, and their affinity and kinetics profiles were determined from the resulting sensorgrams. Kinetics were run in duplicate over three independent mIFN-γ surfaces (n = 6). Representative sensorgrams of concentration series showing the experimental data as well as the fit binding curves calculated using BIAevaluation software are shown. The Δ3 sensorgram is typical of those observed for inactive truncation mutants, and F261A is typical of sensorgrams for the alanine scanning mutants. Constructs for which a very low or background response could be generated have very low to no affinity for mIFN-γ.
FIG. 5.
FIG. 5.
Bioactivity of IFN-γBP alanine scanning and truncation mutants. Wild-type and C-terminal alanine scanning and truncation mutants of the ECTV IFN-γBP were tested for their ability to neutralize the antiviral effects of IFN-γ. mIFN-γ was preincubated with 0.1-μm-filtered IFN-γBP followed by a 24-h incubation with L929 monolayers. Cells were infected with VSV for 48 h and stained with crystal violet to assess viability. The wild type and all C-terminal alanine mutants were able to effectively neutralize the antiviral effects of IFN-γ. C-terminal truncation (Δ8) or disruption of the local secondary structure (F261P) resulted in an inability to block the antiviral effects of IFN-γ.
FIG. 6.
FIG. 6.
The IFN-γBP C terminus mediates noncovalent oligomerization, as demonstrated in a gel filtration analysis of IFN-γBP mutants. A 2.5-μg aliquot of each construct was injected in a volume of 500 μl over a calibrated Superdex 200 HR column (GE Healthcare, Piscataway, NJ) in 100 mM Tris pH 7.0, 500 mM NaCl, 0.1% Triton X-100. Fractions of 0.5 ml were analyzed by reducing SDS-PAGE and Western blotting for IFN-γBP. Migration of each construct relative to gel filtration standards (Bio-Rad) is indicated. IFN-γ binding activity correlates with migration as a complex with a median size of 250 kDa, whereas the nonactive construct migrates as smaller complexes with a median size of 80 kDa, likely representing covalent dimers. Full-length proteins were resistant to reduction with DTT, whereas truncated IFN-γBP could be reduced to a peak with a median size of 40 kDa, consistent with a monomeric form of the protein. SM, starting material.
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